The present application relates to a fluorescence intensity compensation method and a fluorescence intensity computing apparatus and, in particular, to a fluorescence intensity control method for accurately computing the intensity of fluorescence emitted from each of a plurality of fluorochromes multiply-labeled on a microparticle.
In order to measure a characteristic of a microparticle, such as a cell, existing apparatuses (e.g., flow cytometers) labels the microparticle using a fluorochrome, emits a laser beam onto the fluorochrome to excite the fluorochrome, and measures the intensity or pattern of the fluorescence emitted from the excited fluorochrome. In recent years, in order to more precisely analyze the characteristic of, for example, a cell, multi-color measurement has been used in which a microparticle is labeled using a plurality of fluorochromes and light beams emitted from the fluorochromes are measured using a plurality of photodetectors (e.g., PDs (photodiodes) or PMTs (photomultipliers)) having different wavelength ranges of received light. In multi-color measurement, in order to detect the fluorescence, one of optical filters is selected for a photodetector in accordance with the fluorescence wavelength of the fluorochrome being in use.
However, the currently used fluorochromes (e.g., FITC (fluorescein isothiocyanate) or PE (phycoerythrin)) have the overlap frequency range in the fluorescence spectrum. Accordingly, in the case where multi-color measurement is conducted using a combination of these fluorochromes, even when the fluorescence emitted from each of the fluorochromes is separated into individual frequency ranges using optical filters, the photodetectors may detect the fluorescence spilled over from an unwanted fluorochrome. If spillover of fluorescence occurs, the fluorescence intensity detected by the photodetector is shifted from the true intensity of fluorescence emitted from a desired fluorochrome. Thus, an error in a measurement arises.
In order to compensate the error in a measurement, fluorescence compensation is performed by subtracting the intensity of spillover fluorescence from the fluorescence intensity measured by the photodetector. The fluorescence compensation is performed by electrically or mathematically compensating a pulse so that the fluorescence intensity measured by the photodetector becomes a true fluorescence intensity of a desired fluorochrome.
A method for mathematically compensating the fluorescence intensity is performed by defining the fluorescence intensities (the detection values) measured by the photodetectors as a vector and applying the inverse matrix of a predetermined spillover matrix to the vector. In this way, a true intensity of the fluorescence emitted from a desired fluorochrome can be computed (refer to FIGS. 3 and 4 and Japanese Unexamined Patent Application Publication No. 2003-83894). The spillover matrix is generated by analyzing the fluorescence wavelength distribution of microparticles each of which is simple-labeled. In the spillover matrix, the fluorescence wavelength distribution of each of the fluorochromes is expressed as a column vector, and the column vectors are arranged. The inverse matrix of the spillover matrix is also referred to as a “compensation matrix”. In FIGS. 3 and 4, the case in which five-color measurement is conducted using five types of fluorochrome (FITC, PE, ECD, PC5, and PC7) and five photodetectors is shown.